Journal: Frontiers in pharmacology

Article Title: Activating Cannabinoid Receptor 2 Protects Against Diabetic Cardiomyopathy Through Autophagy Induction.

doi: 10.3389/fphar.2018.01292

Figure Lengend Snippet: FIGURE 2 | Administration of HU308 enhances cardiac autophagy in heart issues from DCM mice. (A) Representative images of the autophagy-related proteins including beclin-1, LC3-II/I, and p62. (B) Quantitative analysis of the autophagy-related proteins (n = 5). ∗∗P < 0.01 vs. Nor group; $$P < 0.01 vs. DCM group. Nor, normal; DCM, diabetic cardiomyopathy.

Article Snippet: The primary antibodies applied were listed as follows: rabbit anti-Beclin-1 monoclonal antibody (1:1000; Cell Signaling Technology, Danvers, MA, United States), rabbit anti-LC3 polyclonal antibody (1:1000; Novus Biologicals, Littleton, CO, United States), rabbit anti-p62 antibody (1:1000; Cell Signaling Technology, Danvers, MA, United States), rabbit anti-adenosine 5′- monophosphate (AMP)-activated protein kinase (AMPK) antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-phosphorylated AMPK antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States) anti-mammalian target of rapamycin rabbit (mTOR) antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-phosphorylated mTOR antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-p70 ribosomal protein S6 kinase (p70S6K) antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States), rabbit antiphosphorylated p70S6K antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States) and mouse anti-β-actin antibody (1:5000, Beyotime Biotechnology, Shanghai, China).

Techniques:

Journal: Frontiers in pharmacology

Article Title: Activating Cannabinoid Receptor 2 Protects Against Diabetic Cardiomyopathy Through Autophagy Induction.

doi: 10.3389/fphar.2018.01292

Figure Lengend Snippet: FIGURE 4 | Administration of HU308 enhances cardiac autophagy in cardiomyocytes under the challenge of HG. Primary murine ventricular cardiomyocytes were obtained from neonatal C57BL/6J mice and challenged with high glucose concentration (HG; 33 mM glucose) for 24 h as hyperglycemia group or medium with normal glucose concentration (Nor; 5.5 mM glucose) as the control group. In certain groups, HU308 (10 µM) was treated to cells at 10 min before suffering HG. PBS in the same volume was administrated as vehicle. (A) Representative images of the autophagy-related proteins including beclin-1, LC3-II/I, and p62. (B) Quantitative analysis of the autophagy-related proteins (n = 5). ∗∗P < 0.01 vs. Nor group; $$P < 0.01 vs. HG group. (C) Representative images of transmission electron microscopy with arrows marking autophagosomes. (D) Quantitative analysis of the number of autophagosomes in murine ventricular cardiomyocytes (n = 5). ∗∗P < 0.01 vs. Nor group; $$P < 0.01 vs. HG group. Nor, normal; DCM, diabetic cardiomyopathy.

Article Snippet: The primary antibodies applied were listed as follows: rabbit anti-Beclin-1 monoclonal antibody (1:1000; Cell Signaling Technology, Danvers, MA, United States), rabbit anti-LC3 polyclonal antibody (1:1000; Novus Biologicals, Littleton, CO, United States), rabbit anti-p62 antibody (1:1000; Cell Signaling Technology, Danvers, MA, United States), rabbit anti-adenosine 5′- monophosphate (AMP)-activated protein kinase (AMPK) antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-phosphorylated AMPK antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States) anti-mammalian target of rapamycin rabbit (mTOR) antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-phosphorylated mTOR antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-p70 ribosomal protein S6 kinase (p70S6K) antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States), rabbit antiphosphorylated p70S6K antibody (1:1000, Cell Signaling Technology, Danvers, MA, United States) and mouse anti-β-actin antibody (1:5000, Beyotime Biotechnology, Shanghai, China).

Techniques: Concentration Assay, Control, Transmission Assay, Electron Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

doi: 10.3390/ijms22041834

Figure Lengend Snippet: Observation of microtubule-associated proteins 1A/1B light chain 3 (LC3) and actin double-stained fluorescence images of rat embryonic fibroblasts (REF) cells. ( A ) Indirect fluorescein isothiocyanate (FITC) immunofluorescence labelling of LC3 using optical microscope (OM) with a green fluorescence filter (see Methods; 400× magnification). ( B ) Indirect rhodamine immunofluorescence labelling of actin viewed through a red fluorescence filter (see Methods; 400× magnification). An enlarged image of the white framed area is shown in the upper corner. ( C ) A merged fluorescence image of LC3 and actin. ( D , E ) Enlarged images of the LC3-rich areas (indicated by yellow arrows in C ). ( F , G ) Enlarged images of the LC3-poor areas (indicated by white arrows in ( C )) showing many filamentous actin bundles. ( H ) A merged image of LC3 and phalloidin-labelled filamentous actin. ( I , J ) Enlarged images of the LC3-rich areas (indicated by yellow arrows in ( H )). ( K , L ) Enlarged images of the LC3-poor areas (indicated by white arrows in ( H )). Scale bars: 50 μm in ( A – C , H ), 10 μm in ( D–G , I–L ).

Article Snippet: The cells were stained with mouse anti-LC3 antibody (MBL, Cat#: M152-3, 1/50) for 30 min, washed twice with PBS twice, and stained with anti-mouse IgG-conjugated 60 nm gold colloids (Cytodiagnostics Inc., Burlington, ON, Canada, Cat #: AC-60-02-05, 1/50) for 30 min. After washing twice, the holder was attached to the acrylic sample holder on the 50 nm SiN film and observed with the SE-ADM system [ , ].

Techniques: Staining, Fluorescence, Immunofluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

doi: 10.3390/ijms22041834

Figure Lengend Snippet: Observation of Atg12 and actin double-stained fluorescence images of REF cells. ( A ) Indirect FITC immunofluorescence labelling of Atg12 using OM with a green fluorescence filter (see Materials and Methods; 400× magnification). Small fluorescence spots near the nucleus are Atg12. ( B ) A fluorescence image of actin filament stained by rhodamine-conjugated phalloidin, viewed through a red fluorescence filter (400× magnification). ( C ) A merged fluorescence image of Atg12 and actin. ( D , E ) Enlarged images of the Atg12-rich areas (indicated by yellow arrows in ( C )). ( F , G ) Enlarged images of the Atg12-poor areas (indicated by white arrows in ( C )). Fluorescence spots of Atg12 are rare near the filamentous actin. ( H , I ) Enlarged images of the LC3 and Atg12 fluorescence spots, respectively. Scale bars: 50 μm in ( A – C ), 10 μm in ( D – I ).

Article Snippet: The cells were stained with mouse anti-LC3 antibody (MBL, Cat#: M152-3, 1/50) for 30 min, washed twice with PBS twice, and stained with anti-mouse IgG-conjugated 60 nm gold colloids (Cytodiagnostics Inc., Burlington, ON, Canada, Cat #: AC-60-02-05, 1/50) for 30 min. After washing twice, the holder was attached to the acrylic sample holder on the 50 nm SiN film and observed with the SE-ADM system [ , ].

Techniques: Staining, Fluorescence, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

doi: 10.3390/ijms22041834

Figure Lengend Snippet: Dielectric images of 4T1E/M3 cells stained with anti-LC3 antibody conjugated to 60 nm colloidal gold particles. ( A ) Dielectric images of the cells stained with anti-LC3 antibody conjugated to 60 nm colloidal gold particles after paraformaldehyde fixation and permeabilization (10,000× magnification, 6 kV electron beam (EB)). ( B ) An enlarged image of the red framed area in ( A ), showing LC3 bound to 60 nm colloidal gold particles. ( C ) A pseudo-color map of ( B ) after intensity inversion. ( D ) A schematic diagram of arc-like structures and colloidal gold particles superimposed on a dielectric image of ( B ). ( E ) A 3D color map of ( C ). ( F ) Another scanning electron-assisted dielectric microscopy (SE-ADM) image of 4T1E/M3 cells (10,000× magnification, 6 kV EB). ( G ) An enlarged image of the red framed area in ( E ). ( H ) A pseudo-color map of ( F ) after intensity inversion. LC3 aligns in a circular manner. ( I ) A 3D color map of ( H ). Scale bars: 1 μm in ( A , F ), 500 nm in ( B – E , G – I ).

Article Snippet: The cells were stained with mouse anti-LC3 antibody (MBL, Cat#: M152-3, 1/50) for 30 min, washed twice with PBS twice, and stained with anti-mouse IgG-conjugated 60 nm gold colloids (Cytodiagnostics Inc., Burlington, ON, Canada, Cat #: AC-60-02-05, 1/50) for 30 min. After washing twice, the holder was attached to the acrylic sample holder on the 50 nm SiN film and observed with the SE-ADM system [ , ].

Techniques: Staining, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

doi: 10.3390/ijms22041834

Figure Lengend Snippet: Dielectric images of REF cells stained with anti-Atg12 antibody conjugated to 60 nm colloidal gold particles. ( A ) A low-magnification dielectric image of the cells stained with anti-Atg12 antibody conjugated to 60 nm colloidal gold particles after fixation and permeabilization (1500× magnification, 6 kV EB). ( B ) A high-magnification image (10,000×) of the red framed area in ( A ), showing Atg12 conjugated to 60 nm colloidal gold particles. ( C ) An enlarged image of the red framed area in ( B ). ( D ) A pseudo-color map of ( C ) after intensity inversion. ( E ) A schematic diagram of meshwork structures and colloidal gold particles superimposed on a dielectric image of ( C ). ( F ) A 3D color map of ( D ). ( G ) Histograms of minimum intervals between pairs of colloidal gold particles. Pairs of particles (183 for LS3 and 300 for Atg12) were randomly selected and the intervals were measured. Minimum intervals between LC3-bound colloids are small and sharply peaked, whereas those between Atg12 are larger and gently distributed. ( H ) A schematic illustration of the structural organization of autophagosomes involving LC3 and Atg12 with actin. Scale bars: 10 μm in ( A ), 1 μm in ( B ), 500 nm in ( C – F ).

Article Snippet: The cells were stained with mouse anti-LC3 antibody (MBL, Cat#: M152-3, 1/50) for 30 min, washed twice with PBS twice, and stained with anti-mouse IgG-conjugated 60 nm gold colloids (Cytodiagnostics Inc., Burlington, ON, Canada, Cat #: AC-60-02-05, 1/50) for 30 min. After washing twice, the holder was attached to the acrylic sample holder on the 50 nm SiN film and observed with the SE-ADM system [ , ].

Techniques: Staining

Journal: Acta pharmacologica Sinica

Article Title: Enhanced lysosomal function is critical for paclitaxel resistance in cancer cells: reversed by artesunate.

doi: 10.1038/s41401-020-0445-z

Figure Lengend Snippet: Fig. 3 Artesunate inhibits the activity of cathepsins in MCF-7 cells. a Western blot detection of cathepsin L in the MCF-7 cells after treatment with artesunate for 24 h. b Western blot detection of cathepsin D in the MCF-7 cells after treatment with artesunate for 24 h. c LysoTracker detection in the MCF-7 cells after treatment with artesunate for 24 h. d Electron microscopy results showing autophagy in the MCF-7 cells treated with artesunate for 24 h. The black arrow indicates autophagosomes, and the red arrow indicates autolysosomes. e Western blot detection of P62 and LC3 in the MCF-7 cells after treatment with artesunate for 24 h in the presence or absence of bafilomycin A1. Actin was used as the loading control. Quantitative analysis was performed with ImageJ software. Values are presented as the means ± SD from three independent experiments. *P < 0.05, ***P < 0.001, and ns P > 0.05 versus the control group.

Article Snippet: The following antibodies were used: anti-PARP antibody (#9532, Cell Signaling Technology), anti-Caspase3 antibody (#9661, Cell Signaling Technology), anti-Cytochrome c antibody (#11940, Cell Signaling Technology), anti-Cathepsin B antibody (#C6243, Sigma), anti-Cathepsin D antibody (#SC-377299, Santa Cruz), anti-Cathepsin L antibody (#ab6314, Abcam), anti-P62 antibody (#P0067, Sigma), anti-LC3 antibody (#NB600-1384, Novus Biologicals), anti-TFEB antibody (#4240, Cell Signaling), anti-LAMP1 antibody (#SAB3500285, Sigma), anti-GAPDH antibody (#ab9484, Abcam), anti-COX IV antibody (#11967,Cell Signaling Technology), and anti-β-Actin antibody (#A5441, Sigma).

Techniques: Activity Assay, Western Blot, Electron Microscopy, Control, Software

Journal: Journal of neuroinflammation

Article Title: Role of macrophage autophagy in postoperative pain and inflammation in mice.

doi: 10.1186/s12974-023-02795-w

Figure Lengend Snippet: Fig. 1 Confirmation of Atg5 gene knockout and autophagy deficiency in macrophages. A Polymerase chain reaction analysis of gene in peritoneal macrophages from wild type (WT), Atg5f/f, and Atg5f/fLysMCre (Atg5 cKO) mice. B Western blot analysis of ATG5 and SQSTM1/ p62 proteins in peritoneal macrophages from Atg5f/f and Atg5f/fLysMCre mice (n = 5 each) cultured for 6 h under energy-depletion. C Densitometric quantification of western blot of ATG5, SQSTM1/p62, and LC3 proteins (n = 3–5 each). D Representative immunofluorescence images of macrophages. E LC3 immuno-staining density of macrophages (n = 3 each). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. cKO conditional knockout; WT wild type

Article Snippet: Macrophages were then stained with Alexa Fluor 635-conjugated rabbit anti-mouse ionized calcium-binding adapter molecule 1 (Iba1, 1:200; 013-26471, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) and Alexa Fluor 488-conjugated rabbit anti-mouse LC3 (1: 200; NB600-1384AF488, Novus Biologicals, Colorado, USA) overnight in the dark at room temperature.

Techniques: Gene Knockout, Polymerase Chain Reaction, Western Blot, Cell Culture, Immunofluorescence, Immunostaining, Knock-Out